1. Field of the Invention
The present invention relates generally to the fields of molecular biology and nucleic acid analysis. More specifically, the present invention relates to a novel method of nucleic acid analysis using tandem hybridization approaches.
2. Description of the Related Art
The emerging field of DNA technology is bringing powerful analytical capabilities in research and clinical laboratories. A prime example is the identification of mutations causing genetic diseases, such as the detection of .DELTA.F508 mutation responsible of cystic fibrosis disease (Riordan et al, Science 245:1066-1073, 1989). Many technical approaches have been devised to search for the presence of one or a few mutations in a single assay. However, it is now evident that many genetic diseases can be caused by a wide variety of mutations in the gene altered, as with cystic fibrosis, where more than 200 different mutations in the CFTR gene have been detected in CF patients (Tsui, Trends Genet. 8:392-398, 1992). Several analytical approaches have recently been proposed to simultaneously investigate multiple mutations in a single assay. For example, fluorescently labeled allele-specific oligonucleotides have been used in the analysis of several mutations by PCR (Heller, In The Polymerase Chain Reaction, Mullis et al, Eds., Birkhausen, Boston, pp. 134-141, 1994) or the ligase chain reaction (Jou et al, Human Mutat. 5:86-93, 1995; Eggerdin, Human Mutat. 5:153-165, 1995). Oligonucleotide hybridization shows promise as a rapid and sensitive method for simultaneous analysis of large numbers of mutations (Conner et al, Proc. Natl. Acad. Sci., USA. 80:278-282, 1983; Southern, International patent application PCT GB 89/00460, 1988; Beattie et al, Clin. Chem. 39:719-722, 1992; Southern et al, Genomics 13:1008-1017, 1992; Maskos & Southern, Nucl. Acids Res. 20:1675-1678, 1992; Mirzabekov, Trends Biotechnol. 12:27-32, 1994; Case-Green et al, In Innovation and Perspectives in Solid Phase Synthesis, Proc. 3rd International Symposium on Solid Phase Synthesis, Epton, Ed., Mayflower Worldwide Ltd., Birmingham, U.K., pp. 77-82, 1994; Pease et al, Proc. Natl. Acad. Sci., U.S.A. 91:5022-5026, 1994; Nikiforov et al, Nucl. Acids Res. 22:4167-4175, 1994; Beattie et al, Molec. Biotechnol. 4:213-225, 1995; Beattie et al, Clin. Chem. 41:700-706, 1995; Parinov et al, Nucl. Acids Res. 24:2998-3004, 1996; Yershov et al, Proc. Natl. Acad. Sci., U.S.A. 93:4913-4918, 1996; Hacia et al, Nature Genetics 14:441-447, 1996; Southern, Trends Genet. 12:110-115, 1996). Various strategies have been described which enable simultaneous analysis of numerous oligonucleotide hybridization reactions. The most common strategy employed to separately analyze the hybridization of numerous oligonucleotide probes to a nucleic acid analyte is to immobilize each oligonucleotide at a specific, addressable site on a surface, then to label the analyte nucleic acid, hybridize it to the oligonucleotide array, and measure the relative quantity of label bound at each position across the array, using a CCD imaging system, a scanning confocal microscope, phosphorimager, exposure of X-ray film, etc. The inverse situation to the use of oligonucleotide arrays to analyze a nucleic acid sample is to immobilize numerous nucleic acid samples in a two-dimensional array, then to analyze the binding of a DNA probe to each of the arrayed analytes. Included in the latter approach are membrane hybridizations using size-separated nucleic acid fragments (as in Southern blots and Northern blots) and slot blots and dot blots in which each analyte is placed onto the membrane at a specific location. In addition, high density arrays of genomic clones, cDNAs, gene-specific amplicons or other PCR products, immobilized onto membranes or onto glass or silicon surfaces, are frequently used in hybridizations with oligonucleotide probes or longer nucleic acid fragments, for genome mapping, genotyping and gene expression profiling. Another approach to multiplex DNA hybridization is to immobilize each DNA probe to microbeads color-coded with a specific "signature" of fluorophores, then to hybridize the analyte nucleic acid labeled with a molecular tag with the bead mixture and analyze the mixture by flow cytometry, using the fluorescent signature to resolve each probe and the molecular tag to quantitate the binding of analyte to each probe (FlowMetrix method of Luminex, Inc.).
Simultaneous hybridization of a DNA sample to numerous oligonucleotide probes attached to a solid support material ("DNA chip," or "genosensor") has been proposed as a powerful research tool in various kinds of DNA sequence analysis, including sequencing by hybridization, scanning for known or unknown mutations in a gene of known nucleotide sequence, genotyping of organisms, and genome mapping (Southern, International patent application PCT GB 89/00460, 1988; Beattie et al, Clin. Chem. 39:719-722, 1992; Southern et al, Genomics 13:1008-1017, 1992; Maskos & Southern, Nucl. Acids Res. 20:1675-1678, 1992; Mirzabekov, Trends Biotechnol. 12:27-32, 1994; Case-Green et al, In Innovation and Perspectives in Solid Phase Synthesis, Proc. 3rd International Symposium on Solid Phase Synthesis, Epton, Ed., Mayflower Worldwide Ltd., Birmingham, U.K., pp. 77-82, 1994; Pease et al, Proc. Natl. Acad. Sci., U.S.A. 91:5022-5026, 1994; Nikiforov et al, Nucl. Acids Res. 22:4167-4175, 1994; Beattie et al, Molec. Biotechnol. 4:213-225, 1995; Beattie et al, Clin. Chem. 41:700-706, 1995; Parinov et al, Nucl. Acids Res. 24:2998-3004, 1996; Yershov et al, Proc. Natl. Acad. Sci., U.S.A. 93:4913-4918, 1996; Hacia et al, Nature Genetics 14:441-447, 1996; Southern, Trends Genet. 12:110-115, 1996; Bains & Smith, J. Theor. Biol. 135:303-307, 1988; Drmanac et al, Genomics 4:114-128, 1989; Khrapko et al, FEBS Lett. 256:118-122, 1989; Khrapko et al, DNA Sequence 1:375-388, 1991; Bains, Genomics 11:294-301, 1991; Fodor et al, Science 251:767-773, 1991; Drmanac & Crkvenjakov, Int. J. Genome Res. 1:59-79, 1992; Drmanac et al, Science 260:1649-1652, 1993; Bains, DNA Sequence 4:143-150, 1993; Meier-Ewert et al, Nature 361:375-376, 1993; Broude et al, Proc. Natl. Acad. Sci., USA 91:3072-3076, 1994; Hoheisel, Trends Genet. 10:79-83, 1994; Drmanac & Drmanac, BioTechniques 17:328-336, 1994; Lamture et al, Nucl. Acids Res. 22:2121-25, 1994; Caetano-Anolles, Nature Biotechnol. 14:1668-1674, 1996; Lockhart et al, Nature Biotechnol. 14:1675-1680, 1996; Milner et al, Nature Biotechnol. 15:537-541, 1997). Despite widespread interest generated about the various multiplex hybridization technologies, several technical challenges remain to be solved before these techniques can reach their full potential and be successfully implemented in a robust fashion. One problem, anticipated from the beginning, is the spontaneous formation of secondary structure in the single stranded target nucleic acid, making certain stretches of target sequence poorly accessible to hybridization (Case-Green et al, In Innovation and Perspectives in Solid Phase Synthesis, Proc. 3rd International Symposium on Solid Phase Synthesis, Epton, Ed., Mayflower Worldwide Ltd., Birmingham, U.K., pp. 77-82, 1994; Beattie et al, Clin. Chem. 41:700-706, 1995; Milner et al, Nature Biotechnol. 15:537-541, 1997). This problem may be especially difficult when short oligonucleotide probes are used, wherein the hybridization temperature is too low to disrupt some regions of intrastrand secondary structure. Many applications of membrane-, chip- or bead-based hybridization technologies, especially those requiring base mismatch discrimination, may require short probes. One strategy for minimizing the secondary or higher order structure in the DNA target is to fragment the target sequence to very small size. However, such cleavage is difficult to control and does not solve the problem in the case of strong hairpin loops occurring within a short target sequence. The strategy of converting the DNA target to RNA (Hacia et al, Nature Genetics 14:441-447, 1996), which forms a more stable duplex structure with short oligodeoxynucleotide probes than the original DNA target, may also minimize the problem of secondary structure. The RNA targets can be cleaved to short pieces using 25 mM MgCl.sub.2 at 95.degree. C. The procedure used to generate RNA targets from DNA samples, however, is rather cumbersome. Similarly, the use of peptide nucleic acid (PNA) probes, which form even more stable duplex structures with DNA than the RNA.cndot.DNA hybrids cited above (Egholm et al, Nature 365:556-568, 1993) may help solve the secondary structure problem. PNA is expensive, however, and the stabilizing effect is not uniform over all sequences, and furthermore, the discrimination against mismatches appears to be sacrificed in some sequences using PNA probes (Weiler et al, Nucl. Acids Res. 25:2792-2799, 1997).
A further inconvenience in hybridization-based nucleic acid analysis is the need to prepare isolated single-stranded target DNA prior to hybridization to surface-immobilized probes, in order to achieve optimal hybridization signals. Various procedures for isolation of single-stranded targets are available, including the use of affinity columns and strand-specific nuclease digestion, but these added steps are costly, time consuming and inconvenient.
An additional inconvenience in array hybridization analysis is the need to label each nucleic acid analyte prior to hybridization to the DNA probe array. A number of techniques are available for introduction of labels or tags into nucleic acid strands, including (i) the enzymatic incorporation of label from .gamma.-labeled ATP into the 5'-terminus of DNA fragments, using polynucleotide kinase; (ii) incorporation of labeled nucleotides into the target nucleic acid by a polymerase in a "nick translation" or "random primer" labeling reaction, in an in vitro transcription reaction, or in a "reverse transcriptase" reaction; and (iii) direct chemical labeling of DNA or RNA, involving covalent reactions which incorporate fluorescent tags, ligands or haptens into the nucleobases. Although these labeling strategies are straight forward and widely practiced, they nevertheless require additional steps, and if a large number of samples need to be analyzed, the additional labeling steps can be time consuming and expensive. Another problem associated with traditional approaches to sample labeling, especially with complex nucleic acid analytes, is the requirement to introduce a sufficient density of label into the analyte nucleic acid, such that the specific fragment being analyzed is likely to contain at least one label. This can be a problem if the analyte is fragmented prior to hybridization. Furthermore, if a complex mixture of nucleic acid fragments is labeled, nonspecific binding of noncomplementary labeled strands to the array and the occurrence of imperfect hybridization (involving mismatched hybrids) can be a significant problem, since a very small fraction of label will be present on the specific fragment that is complementary to any given immobilized probe.
Another limitation to sequence-targeted nucleic acid analysis by oligonucleotide array hybridization, especially problematic when short oligonucleotide probes are used or when nucleic acids of high genetic complexity are analyzed, is that more than one complementary sequence may exist within the nucleic acid analyte for any given oligonucleotide probe, making it difficult to target the analysis to unique sites.
It is well known that in oligonucleotide hybridization, base mismatches at the terminal positions of the probe are difficult to discriminate against, while multiple mismatches are readily discriminated against and single internal mismatches are discriminated against to an intermediate extent and sometimes poorly. Furthermore, short oligonucleotide hybridization is known to be highly influenced by base composition, nearest neighbor and probe length, so that a large amount of experimentation is required in order to identify oligonucleotide probes that yield reliable and interpretable hybridization results, and furthermore, if an extensive oligonucleotide array is used, the numerous probes must be designed to form duplex structures (hybrids) of very similar thermal stability.
Stabilization of short duplex structures by base stacking interactions between tandemly hybridized (contiguously stacked) oligonucleotides has been described (Parinov et al, Nucl. Acids Res. 24:2998-3004, 1996; Yershov et al, Proc. Natl. Acad. Sci., U.S.A. 93:4913-4918, 1996; Khrapko et al, FEBS Lett. 256:118-122, 1989; Khrapko et al, DNA Sequence 1:375-388, 1991; Kieleczawa et al, Science 258:1787-1791, 1992; Kotler et al, Proc. Natl. Acad. Sci., U.S.A. 90:4241-4245, 1993; Kaczorowski & Szybalski, Anal. Biochem. 221:127-135, 1994; Kaczorowski & Szybalski, Gene 179:189-193, 1996; Lodhi & McCombie, Genome Res. 6:10-18, 1996; Johnson et al, Anal. Biochem. 241:228-237, 1996). Mutual stabilization of tandemly hybridized oligonucleotides was exploited in the use of a library of all 4096 hexamer primers to obtain readable dideoxy sequencing gels, wherein contiguous strings of three hexamers were annealed to the sequencing template, yielding a priming sequence of 18 bases with or without ligation (Kieleczawa et al, Science 258:1787-1791, 1992; Kotler et al, Proc. Natl. Acad. Sci., U.S.A. 90:4241-4245, 1993; Kaczorowski & Szybalski, Anal. Biochem. 221:127-135, 1994; Kaczorowski & Szybalski, Gene 179:189-193, 1996; Lodhi & McCombie, Genome Res. 6:10-18, 1996; Johnson et al, Anal. Biochem. 241:228-237, 1996). The Mirzabekov laboratory has shown that such contiguous stacking hybridization may be a viable strategy to resolve sequence ambiguities in sequencing by hybridization (Parinov et al, Nucl. Acids Res. 24:2998-3004, 1996; Khrapko et al, FEBS Lett. 256:118-122, 1989; Khrapko et al, DNA Sequence 1:375-388, 1991) and to identify specific point mutations (Yershov et al, Proc. Natl. Acad. Sci., U.S.A. 93:4913-4918, 1996). The contiguous stacking hybridization strategies taught by the Mirzabekov group, however, require multiple rounds of hybridization. Furthermore, these previous examples of stabilization of short oligonucleotide hybridization through contiguous stacking interactions were observed in hybridization reactions carried out in solution (Kieleczawa et al, Science 258:1787-1791, 1992; Kotler et al, Proc. Natl. Acad. Sci., U.S.A. 90:4241-4245, 1993; Kaczorowski & Szybalski, Anal. Biochem. 221:127-135, 1994; Kaczorowski & Szybalski, Gene 179:189-193, 1996; Lodhi & McCombie, Genome Res. 6:10-18, 1996; Johnson et al, Anal. Biochem. 241:228-237, 1996) or within a polyacrylamide gel matrix (Parinov et al, Nucl. Acids Res. 24:2998-3004, 1996; Yershov et al, Proc. Natl. Acad. Sci., U.S.A. 93:4913-4918, 1996; Khrapko et al, FEBS Lett. 256:118-122, 1989; Khrapko et al, DNA Sequence 1:375-388, 1991). The applicability of stacking hybridization has not been heretofore explored in the environment of a solid surface. Finally, the tandem or contiguous hybridization strategies of the prior art are applicable to analysis of nucleic acid sequences of limited genetic complexity, whereby primers or probes bind to a single unique site within the nucleic acid analyte. Thus, the tandem hybridization approaches of the prior art are inoperable in the analysis of extensive nucleic acid targets, such as complex mixtures of PCR fragments, expressed sequences or total genomes.
Due to deficiencies of the prior art there is a need for improved methods for analysis of numerous known mutations or DNA sequence polymorphisms, using short oligonucleotide probes immobilized on a solid surface. There is also a need for technologies that minimize the influence of probe length and sequence in short oligonucleotide hybridization analysis. There is furthermore a need for improved techniques for analysis of nucleic acid samples of high genetic complexity, using sequence-targeted oligonucleotide array hybridization. Also, there is a need for improved profiling of gene expression using numerous oligonucleotide probes targeted to mRNA species. Moreover, there is a need for more efficient identification of species, strains and individuals using DNA probe arrays designed to hybridize with numerous unique nucleotide sequences. There is in addition a need to adapt oligonucleotide array hybridization to directly analyze nucleic acid samples without the use of additional steps of target sequence amplification, single strand isolation and labeling. The present invention provides a simple, versatile strategy to overcome a variety of technical limitations associated with the prior art.